Title of Project: Endosome to Golgi Retrograde Recycling in C. elegans Background: Cells and tissues establish and maintain their unique architectures in large part through the tight regulation of protein and membrane transport. Key aspects of this process include the decision to recycle or degrade a given molecule, particularly signaling and adhesion molecules, a process that goes awry in many cancers. After endocytosis lipids and trans membrane cargo enter the early endosome for sorting. Cargo may be directed to degrade via targeting to the lysosome, recycled to the PM directly or through the recycling endosome, or sorted for delivery to the Golgi via the retrograde route. A number of observations hint that these routes compete with each other for various cargo, and that the decision to recycle or degrade is a key point of regulation. Our focus is on the much underappreciated branch point between the retrograde route and the degradative route to the lysosome. It has been suggested that competition between these two routes is mediated by microdomains that form on the early endosome and multivesicular body. The clathrin/HGRS-1(Hrs)/ESCRT complexes form one microdomain that promotes degradation via the lysosome, and the retromer/RME-8 complex forms a competing microdomain and promotes the retrograde pathway, recycling cargo from the endosome to the Golgi. The goal of our proposal is to better understand the molecular mechanisms controlling the decision of cargo to traffic via the retrograde or degradative pathways, as well as to find new retrograde components. Objective/Hypothesis: Our lab has recently made advances in our understanding of the molecular mechanism of this competition between the clathrin/HGRS-1(Hrs)/ESCRT and RME- 8/retromer microdomains. Our data suggest that SNX-1, a retromer component, potentiates the binding of RME-8 to the chaperone HSP-1(Hsc70), an interaction that antagonizes the buildup of endosomal clathrin and the associated degradative subdomain via clathrin uncoating activity. Furthermore preliminary evidence suggests that HGRS-1(Hrs) directly competes with this clathrin uncoating process potentially by titrating SNX-1 away from RME-8, leaving clathrin free to build up and promote lysosomal degradation. We aim to test this microdomain maintenance hypothesis, as well as dramatically extend our knowledge of the retrograde pathway in a multicellular organism by large-scale RNAi-based screening. Specific Aims: (1) To determine the functional consequence of SNX-1/RME-8 interaction (2) Distinguish between two models to explain the HGRS-1(Hrs)/SNX-1 interaction, especially as it relates to RME-8/HSP-1(Hsc70) mediated endosomal sorting and (3) To identify novel components of the retrograde sorting pathway. Study design: We propose to analyze our competition model using a combination of in vivo rescue experiments with interaction impaired mutants of RME-8, epistasis analysis, and Fvrster resonance energy transfer (FRET) analysis. Additionally we propose a simple yet powerful large scale RNAi screen to identify novel retrograde pathway components. Significance: All of the components of retrograde and lysosomal sorting we are studying are conserved in mammals. Defects in retrograde transport are linked to late-onset Alzheimer's disease, E-cadherin mis-localization, defects in the generation of Wnt signaling gradients, and defects in the clearance of dead apoptotic cells. Additionally the retrograde sorting route is the mode of entry for Shiga Toxin. We believe that a better understanding the molecular mechanism that target MIG-14 to its proper location within the cell, as well as identifying new genes that have a role in this process, will help us to understand the underlying molecular events leading a vast array of disease states. PUBLIC HEALTH RELEVANCE: Endocytosis and endosomal sorting involves the uptake of lipids and proteins from the cell surface followed by their proper distribution in the cell. This trafficking is critical for diverse cellular functions such as signaling, cell-cell junction maintenance, nutrient uptake, and development. Defects in transport are linked to late- onset Alzheimer's disease, defects in cellular polarity (knowing its top from its bottom) and defects in the clearance of dead cells.